U.S. patent number 7,507,588 [Application Number 11/404,348] was granted by the patent office on 2009-03-24 for multiplex microparticle system.
This patent grant is currently assigned to Becton, Dickinson and Company. Invention is credited to Majid Mehrpouyan, Diether J. Recktenwald, Rudolf Varro.
United States Patent |
7,507,588 |
Mehrpouyan , et al. |
March 24, 2009 |
Multiplex microparticle system
Abstract
Arrays of microparticle populations, each population labeled
with a single fluorescent dye, are provided for use in multiplex
assays. The populations form a virtual multidimensional array
wherein each microparticle is identified by fluorescence intensity
in two different fluorescence detection channels. The arrays are
useful in a variety of assays, including multiplex, multi-analyte
assays for the simultaneous detection of two or more analytes by,
for example, flow cytometry, and a labeling reagents in, for
example, microscopy. The use of singly-dyed microparticles to form
multidimensional arrays greatly simplifies the creation of
multiplex assays.
Inventors: |
Mehrpouyan; Majid (Gilroy,
CA), Recktenwald; Diether J. (San Jose, CA), Varro;
Rudolf (Mountain View, CA) |
Assignee: |
Becton, Dickinson and Company
(Franklin Lakes, NJ)
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Family
ID: |
37215231 |
Appl.
No.: |
11/404,348 |
Filed: |
April 14, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060240411 A1 |
Oct 26, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60673471 |
Apr 20, 2005 |
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Current U.S.
Class: |
436/518;
435/287.2; 435/4; 435/6.1; 435/6.11; 435/7.1; 435/7.2; 436/164;
436/172; 436/44; 436/46; 436/517; 436/523; 436/524; 436/525;
436/526; 436/527; 436/528; 436/529; 436/530; 436/531; 436/546 |
Current CPC
Class: |
G01N
33/54313 (20130101); G01N 33/582 (20130101); G01N
33/585 (20130101); Y10T 436/110833 (20150115); Y10T
436/101666 (20150115); Y10T 436/112499 (20150115); Y10S
435/973 (20130101) |
Current International
Class: |
G01N
33/543 (20060101) |
Field of
Search: |
;435/6,7.1,4,7.2,287.2,973
;436/517,523-535,546,10,15,164,172,44,46,518 |
References Cited
[Referenced By]
U.S. Patent Documents
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4342739 |
August 1982 |
Kakimi et al. |
4717655 |
January 1988 |
Fulwyler et al. |
5073498 |
December 1991 |
Schwartz et al. |
5326692 |
July 1994 |
Brinkley et al. |
5369036 |
November 1994 |
Mercolino et al. |
5716855 |
February 1998 |
Lerner et al. |
5981180 |
November 1999 |
Chandler et al. |
6023540 |
February 2000 |
Walt et al. |
6268222 |
July 2001 |
Chandler et al. |
6514295 |
February 2003 |
Chandler et al. |
6524793 |
February 2003 |
Chandler et al. |
6528165 |
March 2003 |
Chandler et al. |
6642062 |
November 2003 |
Kauvar et al. |
6680211 |
January 2004 |
Barbera-Guillem et al. |
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Foreign Patent Documents
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1 248 873 |
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Jan 1989 |
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CA |
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0 296 136 |
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Dec 1988 |
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EP |
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1 561 042 |
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Feb 1980 |
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GB |
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Other References
Fulton, Jerrold et al., "Advanced multiplex analysis with the
FlowMetrixTM system," Clinical Chemistry, vol. 43 (9): 1749-1756
(1997). cited by other .
Cook, E. B. et al., "Simultaneous measurement of six cytokines in a
single sample of human tears using microparticle-based flow
cytometry: allergics vs. non-allergics ," Journal of Immunology
Methods, vol. 254: 109-118 (2001). cited by other .
Chen Roy et al: "Simultaneous quantification of six human cytokines
in a single sample using microparticles-based flow cytometric
technology" Sep. 1999, Clinical Chemistry, vol. 45, Nr. 9, pp.
1693-1695, Oak Ridge Conference, San Jose, California, USA. cited
by other.
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Primary Examiner: Gabel; Gailene R
Attorney, Agent or Firm: Petry; Douglas A.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional application
No. 60/673,471, filed Apr. 20, 2005, the disclosure of which is
incorporated herein by reference.
Claims
We claim:
1. An array of populations of microparticles, comprising a
plurality of sets, wherein each set contains a plurality of
populations of microparticles, wherein microparticle populations
within a set are labeled with different levels of a single
fluorophore such that each population exhibits a measurably
distinct fluorescence intensity; wherein microparticles in
different sets are labeled with different fluorophores; wherein all
of said fluorophores have overlapping emission spectra and emit
light that is detectable using a single pair of detection channels,
and the relative emission in each of the two detection channels is
distinguishably distinct between different fluorophores; wherein
each of said microparticle populations is distinguishable by its
fluoresence emissions in said pair of detection channels; and
wherein said array comprises at least 3 sets of microparticle
populations.
2. The array of claim 1, wherein said array comprises at least 4
sets of microparticle populations.
3. An array of microparticles for detecting multiple analytes in a
sample, comprising: a) an array of microparticles of claim 2; b) a
plurality of analyte-specific reagents, wherein reagents of the
same specificity are coupled to microparticles in the same
population, and reagents of different specificity are coupled to
microparticles in different populations.
4. The array of claim 1, wherein said array comprises at least 5
sets of microparticle populations.
5. An array of microparticles for detecting multiple analytes in a
sample, comprising: a) an array of microparticles of claim 4; b) a
plurality of analyte-specific reagents, wherein reagents of the
same specificity are coupled to microparticles in the same
population, and reagents of different specificity are coupled to
microparticles in different populations.
6. The array of claim 1, wherein said array comprises at least 20
populations of microparticles.
7. An array of microparticles for detecting multiple analytes in a
sample, comprising: a) an array of microparticles of claim 6; b) a
plurality of analyte-specific reagents, wherein reagents of the
same specificity are coupled to microparticles in the same
population, and reagents of different specificity are coupled to
microparticles in different populations.
8. The array of claim 1, wherein said array comprises at least 30
populations of micropanicles.
9. An array of microparticles for detecting multiple analytes in a
sample, comprising: a) an array of microparticles of claim 8; b) a
plurality of analyte-specific reagents, wherein reagents of the
same specificity are coupled to microparticles in the same
population, and reagents of different specificity are coupled to
microparticles in different populations.
10. An array of microparticles for detecting multiple analytes in a
sample, comprising: a) an array of microparticles of claim 1; b) a
plurality of analyte-specific reagents, wherein reagents of the
same specificity are coupled to microparticles in the same
population, and reagents of different specificity are coupled to
microparticles in different populations.
11. The array of claim 10, wherein at least one of said
analyte-specific reagents is an antibody that binds specifically to
an analyte that is an antigen, or at least one of said
analyte-specific reactants is an antigen that binds specifically to
an analyte that is an antibody.
12. The array of claim 10, wherein said analytes are selected from
the set consisting of a protein, peptide, hormone, happen, antigen,
antibody, receptor, enzyme, nucleic acid, polysaccaride, chemical,
polymer, pathogen, toxin, organic drug, inorganic drug, cell,
tissue, microorganism, virus, bacteria, fungi, algae, parasite,
allergen, pollutant, or a combination thereof.
Description
BACKGROUND OF THE INVENTION.
1. Field of the Invention
The present invention relates to arrays of labeled microparticles.
Such arrays are particularly useful in multiplex assays, such as
biological detection assays and, more particularly, in the fields
of flow cytometry and fluorescence microscopy.
2. Description of Related Art
Flow cytometers are well known analytical tools that enable the
characterization of particles on the basis of light scatter and
particle fluorescence. In a flow cytometer, particles are
individually analyzed by exposing each particle to an excitation
light, typically one or more lasers, and the light scattering and
fluorescence properties of the particles are measured. Particles,
such as molecules, analyte-bound beads, individual cells, or
subcomponents thereof, typically are labeled with one or more
spectrally distinct fluorescent dyes, and detection is carried out
using a multiplicity of photodetectors, one for each distinct dye
to be detected. Flow cytometers are commercially available from,
for example, BD Biosciences (San Jose, Calif.).
Early in the development of flow cytometry, it was recognized that
various types of ligand binding assays could be carried out using
microparticles (beads) coated with one member of a binding pair.
For example, immunoassays can be carried out in a sandwich
hybridization assay format using beads coated with an
analyte-specific binding agent, such as a monoclonal antibody
((mAb), as a capture reagent, and a second analyte-specific binding
agent, again typically a mAb, labeled with a fluorophore as a
reporter reagent. The coated beads and reporters are incubated with
a sample containing (or suspected of containing) the analyte of
interest to allow for the formation of bead-analyte-reporter
complexes. Analysis by flow cytometry enables both detecting the
presence of bead-analyte-reporter complexes and simultaneously
measuring the amount of reporter fluorescence associated with the
complex as a quantitative measure of the analyte present in the
sample.
It was also recognized early in the development of flow cytometry
that the simultaneous analysis of multiple analytes in a sample
could be carried out using a set of distinguishable beads, each
type of bead coated with a unique analyte-specific binding agent.
The bead set and fluorescently labeled reporter reagents, one for
each species of analyte to be detected, are incubated with a sample
containing the analytes of interest to allow for the formation of
bead-analyte-reporter complexes for each analyte present, and the
resulting complexes are analyzed by flow cytometry to identify and,
optionally, quantify the analytes present in the sample. Because
the identity of the analyte bound to the complex is indicated by
the identity of the bead, multiple analytes can be simultaneously
detected using the same fluorophore for all reporter reagents. A
number of methods of making and using sets of distinguishable
microparticles have been described in the literature.
UK Patent No. 1 561 042, published Feb. 13, 1980, and Fulwyler and
McHugh, 1990, Methods in Cell Biology 33:613-629, describe the use
of multiple microparticles distinguished by size, wherein each size
microparticle is coated with a different target-specific
antibody.
Tripatzis, European Patent No. 0 126,450, published Nov. 28, 1984
(see also corresponding Canadian Patent 1 248 873), describes
multi-dimensional arrays of microparticles formed by labeling
microparticles with two or more fluorescent dyes at varying
concentrations. Microparticles in the array are uniquely identified
by the levels of fluorescence dyes. Tripatzis describes the use of
such arrays for the simultaneous detection a large numbers of
analytes in a sample by flow cytometry, and, further, describes
their use as labels in microscopy.
U.S. Pat. Nos. 4,499,052 and 4,717,655, Entitled: "Method and
Apparatus for Distinguishing Multiple Subpopulations of Cells",
issued Feb. 12, 1985, and Jan. 5, 1988, respectively, describe the
use of microparticles distinguishably labeled with two different
dyes, wherein the microparticles are identified by separately
measuring the fluorescence intensity of each of the dyes.
Both one-dimensional and two-dimensional arrays for the
simultaneous analysis of multiple analytes by flow cytometry are
available commercially. Examples of one-dimensional arrays of
singly dyed beads distinguishable by the level of fluorescence
intensity include the BD.TM. Cytometric Bead Array (CBA) (BD
Biosciences, San Jose, Calif.) and Cyto-Plex.TM. Flow Cytometry
microspheres (Duke Scientific, Palo Alto, Calif.). An example of a
two-dimensional array of beads distinguishable by a combination of
fluorescence intensity (five levels) and size (two sizes) is the
QuantumPlex.TM. microspheres (Bangs Laboratories, Fisher, Ind.). An
example of a two-dimensional array of doubly-dyed beads
distinguishable by the levels of fluorescence of each of the two
dyes is described in McDade and Fulton, April 1997, Medical Device
& Diagnostic Industry; and Fulton et al., 1997, Clinical
Chemistry 43(9):1749-1756.
Each of the microparticle arrays described above has disadvantages
that limit their utility. Arrays based on different size
microparticles are problematical because the amount of capture
reagent that can be bound to a microparticle, which affects the
sensitivity and dynamic range of the assay, is dependent on the
particle size. Thus, to obtain uniform assay performance for all
analytes, it is desirable to use microparticles of uniform size.
One-dimensional arrays based on differences in the fluorescent
intensity of a single dye typically are limited to about 10
different microparticle populations. Although useful for a wide
range of assays, it is desirable to have more distinct
microparticle populations to enable the simultaneous detection of
greater numbers of analytes. Two-dimensional arrays based on
differences in the fluorescence intensities of two distinct dyes
enable much larger arrays, but are significantly more difficult to
manufacture, and increase the difficulty in subsequent data
analysis.
SUMMARY OF THE INVENTION
The present invention relates to multidimensional arrays formed
from populations of singly dyed microparticles. The use of singly
dyed microparticles to form multidimensional arrays greatly
simplifies the creation of multiplex assays, yet still provides
most of the advantages arrays formed from multiply dyed
microparticles.
Multidimensional arrays of the present invention contain a
plurality of sets of microparticle populations, wherein different
populations within a set are labeled, using the same fluorophore,
at a plurality of discrete fluorescence levels, and populations in
different sets are labeled with different fluorophores, wherein the
emission of each fluorophore, as measured in the same two detection
channels, exhibits distinct relative amounts of emission in the two
detection channels. In preferred embodiments, the array contains at
least three sets of microparticle populations, more preferably at
least four, and more preferably at least five.
The present invention is based on the surprising discovery that by
using spectrally similar fluorophores having overlapping emission
spectra, practical multidimensional arrays can be created from a
plurality of one-dimensional arrays, each based on differences in
the fluorescent intensity of a single fluorophore, and that such
arrays created from three or more one-dimensional arrays can be
analyzed using two detection channels. The overlapping emission
spectra of the fluorophores enable the detection of emission from
each of the fluorophores using the same two detection channels.
The fluorescence properties of the multidimensional array of the
invention enable the identification of the microparticles in each
population by exposing the array to excitation light and measuring
the fluorescence of each microparticle in each of the two detection
channels. The resulting fluorescence data can be plotted in a
two-dimensional dot-plot, plotting intensity of the two detection
channels on the two axes, as is routinely used in flow cytometry.
Each population will appear as cluster uniquely positioned in the
two-dimensional dot plot.
The breadth of the emission spectrum of a typical fluorophore is
normally regarded as an undesirable property. For example, in flow
cytometry, fluorescent dyes are selected where possible such that
the emission spectra overlap minimally, and different detector
channels are used to detect different dyes. To maximize detection
sensitivity, each detector channel is selected such that, as much
as possible, it corresponds to the emission maximum of the single
dye that it is intended to detect. Emission detected from the other
dyes, caused by the breadth of the other dye's emission spectrum,
often referred to as "spillover" or "crosstalk", is undesirable and
interferes with the independent measurement of dye fluorescence in
these previously described methods.
In contrast, the present invention makes use of the breadth of the
emission spectrum, and the resulting emission in two detection
channels, to distinguish the fluorophores. The emission spectra of
different fluorophores having similar emission peaks will overlap
each of the two channels to differing degrees and, consequently,
will exhibit different relative emission in the two channels. Thus,
both microparticle populations labeled with different dyes (i.e.,
in different sets) and microparticle populations labeled with
different amount of the same dye (i.e., in the same set) can
distinguished by the different emission intensities in the two
detector channels.
The microparticle arrays of the present invention can be used
essentially in any application in which multiplex particle arrays
are used or are useful, including applications in which the
microparticles are used a solid substrates for ligand binding
assays or as labeling reagents. A preferred use of the arrays is to
implement multiplex binding assays for the simultaneous detection
of two or more analytes using, for example, flow cytometry or
microscopy. For use in such assays, the microparticles are coated
with analyte-specific reagents such that microparticles within a
population are coated with reagents having the same known
specificity and microparticles in different populations are coated
with reagents having different specificities. The identity of the
microparticle populations, determined from the microparticle
fluorescence measured in the two detection channels, enables
identification of the analyte bound to the microparticle through
the analyte-specific reagent. One skilled in the art will
understand that detection can be carried out using any of a number
of different assay formats, including sandwich hybridization
formats and competitive assay formats.
Also comprehended by this disclosure are compositions and kits
which include a multidimensional array disclosed herein.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the overlapping emission spectra of multiple dyes, as
used in the arrays described in Examples 2 and 3.
FIG. 2 shows a dot-plot of an array containing 29 distinct
populations of beads, as described in Example 2.
FIG. 3 shows a dot-pot of an array containing 32 distinct
populations of beads as described in Example 3.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions are provided for clarity. Unless
otherwise indicated, all terms are used as is common in the art.
All reference cited herein, both supra and infra, are incorporated
herein by reference.
As used herein, the terms "microparticles", "microbeads", or
"beads" are used interchangeably. These terms refer to small
particles with a diameter in the nanometer to micrometer range,
typically about 0.01 to 1,000 .mu.m in diameter, preferably about
0.1 to 100 .mu.m, more preferably about 1 to 100 .mu.m, and, for
use in flow cytometry, typically about 1 to 10 .mu.m.
Microparticles can be of any shape, but typically are approximately
spherical ("microspheres").
Microparticles serve as solid supports or substrates to which other
materials, such as target-specific reagents, reactants, and labels,
can be coupled. Microparticles can be made of any appropriate
material (or combinations thereof), including, but not limited to
polymers such as polystyrene; polystyrene which contains other
co-polymers such as divinylbenzene; polymethylmethacrylate (PMMA);
polyvinyltoluene (PVT); copolymers such as styrene/butadiene,
styrene/vinyltoluene; latex; or other materials, such as silica
(e.g., SiO.sub.2).
Microparticles suitable for use in the present invention are well
known in the art and commercially available from a number of
sources. Unstained microspheres in a variety of sizes and polymer
compositions that are suitable for the preparation of fluorescent
microparticles of the invention are available from a variety of
sources, including: Bangs Laboratories (Carmel, Ind.), Interfacial
Dynamics Corporation (Portland, Oreg.), Dynal (Great Neck, N.Y.),
Polysciences (Warrington, Pa.), Seradyne (Indianapolis, Ind.),
Magsphere (Pasadena, Calif.), Duke Scientific Corporation (Palo
Alto, Calif.), Spherotech Inc. (Libertyville, Ill.) and
Rhone-Poulenc (Paris, France). Chemical monomers for preparation of
microspheres are available from numerous is sources.
As used herein, "microparticle population" refers to a group of
microparticles that possess essentially the same optical properties
with respect to the parameters to be measured, such as synthesized
microparticles that, within practical manufacturing tolerances, are
of the same size, shape, composition, and are labeled with the same
kind and amount of dye molecules. For example, unlabeled
microparticles, microparticles labeled with a first dye at a first
concentration, microparticles labeled with the first dye at a
second concentration, and microparticles beads labeled with a
second dye at the third concentration could constitute four
distinct bead populations.
The microparticle arrays of the present invention are detected
using instruments that have the ability to detect fluorescence
light emitted in defined frequency ranges, referred to as "detector
channels" or "detection channels". Typically, such instruments
contain multiple photodetectors, such as photomultiplier tubes or
photodiodes, and the range of wavelengths detected by each
photodetector is determined by the use of frequency-dependent
filters, dichroic mirrors, or other dispersive elements, as is well
known in the art. Alternatively, the same detector can be used for
multiple frequency ranges by changing the dispersive elements
during analysis, as is typical in fluorescence microscopy.
For identification of microparticles in the arrays of the present
invention, two detector channels are used that are close enough
that portions of the emission spectrum of each dye falls within
each channel. The detector channels can be non-overlapping channels
or partially overlapping. The selection of dyes and appropriate
corresponding detection channels is well known and within the
ability of one of skill in the art.
Choice of the detection channels will depend on the application and
the instrumentation used. For example, for use in a flow cytometer
in which two channels are used to detect microparticle fluorescence
and a third is used to detect reporter fluorescence, it is
advantageous to minimize the spillover of the microparticle
emissions into the reporter channel. Thus, the channels are
selected such that the two channels used for detecting the
microparticle emission are spectrally separated from the reporter
channel, within the constraints imposed by the instrument and dyes
available. Again, the selection of compatible dyes and channels is
well known and within the ability of one of skill in the art.
The term "analyte" is used herein broadly to refer to any substance
to be analyzed, detected, measured, or labeled. Examples of
analytes include, but are not limited to: proteins, peptides,
hormones, haptens, antigens, antibodies, receptors, enzymes,
nucleic acids, polysaccarides, chemicals, polymers, pathogens,
toxins, organic drugs, inorganic drugs, cells, tissues,
microorganisms, viruses, bacteria, fungi, algae, parasites,
allergens, pollutants and combinations thereof. It will be
understood that detection of, for example, a cell, is typically
carried out by detecting a particular component, such as a
cell-surface molecule, and that both the component and the bacteria
as a whole can be described as the analyte.
As used herein an "analyte-specific reagent" or "target-specific
reagent" broadly encompasses any reagent that preferentially binds
to an analyte or target of interest, relative to other analytes
potentially present in a sample. A target (analyte) and
target-specific (analyte-specific) reagent are members of a binding
pair, and either member of the pair can be used as the
target-specific reagent in order to selectively bind to the other
member of the pair. Examples of target and target-specific reagent
pairs include, but are not limited to, antigen and antigen-specific
antibody; hormone and hormone receptor; hapten and anti-hapten;
biotin and avidin or steptavidin; enzyme and enzyme cofactor; and
lectin and specific carbohydrate.
Preferred target-specific reagents are antibodies or fragments
thereof that include an antigen binding site that specifically
binds (immunoreacts with) an antigen.
I. Microparticle Array
The array of present invention is comprised of populations of
microparticles, wherein each microparticle is labeled with a single
fluorescent dye. The array consists of a plurality of sets of
microparticle populations, wherein each set contains a plurality of
microparticle populations. Microparticle populations within a set
are labeled, using the same fluorophore, such that each population
exhibits a measurably distinct mean fluorescence intensity.
Microparticle populations in different sets are labeled with
different fluorescent dyes, wherein all of the fluorescent dyes can
be excited by the same excitation light, the emission spectra of
each dye is detectable using the same two detection channels, and
the relative amount of emissions in each of the two detection
channels is distinguishably distinct between different dyes.
The microparticles in the array are detected and uniquely
identified by exposing the microparticles to excitation light and
measuring the fluorescence of each microparticle in each of the two
detection channels. The excitation light may be from one or more
light sources and may be either narrow or broadband. Examples of
excitation light sources include lasers, light emitting diodes, and
arc lamps. Fluorescence emitted in detection channels used to
identify the microparticles may be measured following excitation
with a single light source, or may be measured separately following
excitation with distinct light sources. If separate excitation
light sources are used to excite the microparticle dyes, the dyes
preferably are selected such that all the dyes used to construct
the array are excitable by each of the excitation light sources
used.
For example, a BD FACSCalibur dual laser flow cytometer (BD
Bioscience, San Jose, Calif.) has 488 nm and 635 nm excitation
lasers that are focused on the flow stream at spatially discrete
regions, and detection optics designed to measure light in three
detection channels, designated FL1, FL2, and FL3, following
excitation by the 488 nm laser, and a fourth detection channel,
designated FL4, following excitation by the 635 nm laser. In a
preferred embodiment described in the examples, FL3 and FL4 are
selected as the two detection channels used to identify the
microparticle populations. Thus, in this embodiment, one channel,
FL3, is measured following excitation by the 488 nm laser and the
second channel, FL4, is measured following excitation by the 635 nm
laser. The selection of dyes and detection channels in this example
was made in view of the configuration of an existing commercial
instrument. Alternatively, a flow cytometer could be configured to
measure emission in both FL3 and FL4following excitation with a
single laser.
The resulting fluorescence data from the microparticles preferably
is analyzed by plotting the fluorescence intensity values on a
two-dimensional dot plot, plotting intensity of the two detection
channels on the two axis, as is routinely used in flow cytometry.
Each population of microparticles will yield a cluster uniquely
positioned in the two-dimensional dot plot. The expected patterns
in the dot-plot can be seen from the following analysis.
Let F.sub.i1 and F.sub.i2 be the mean fluorescence measurements of
a ith dyed population in fluorescence channels 1 and 2,
respectively, and R.sub.i=F.sub.i2/F.sub.i1 be the ratio of
intensities for the ith population. Because the relative emission
in the two channels is a property of the fluorophore's emission
spectrum, the ratio of the intensities, for each dye, is a
constant. Thus, in a linear.times.linear dot-plot in which the mean
fluorescence intensities in channels 2 and 1 are plotted on the
ordinate and abscissa, respectively, microparticle populations dyed
with different amounts of the same fluorophore (i.e., populations
within the same set) will fall on the line F.sub.i
2=R.sub.iF.sub.i1, wherein the slope is equal to R.sub.i, the ratio
of emissions in the two channels. Populations in different sets
will fall on lines having different slopes (different ratios of
emissions in the two channels) and will display in different
regions of the dot-plot.
Flourescent intensity data obtained by flow cytometry typically is
plotted using log-transformed data. Using the transformed values,
log(F.sub.i2)=log(R.sub.i)+log(F.sub.i1). Thus, on a log.times.log
dot-plot, populations in all sets are expected to fall on lines
having the same slope, but different "y-intercepts". FIGS. 2 and 3
show data presented on log.times.log dot-plots.
II. Fluorophores
Fluorescent dyes (fluorophores) suitable for use in the present
invention can be selected from any of the many dyes suitable for
use in imaging applications (e.g., flow cytometry). A large number
of dyes are commercially available from a variety of sources, such
as, for example, Molecular Probes (Eugene, Oreg.) and Exciton
(Dayton, Ohio), that provide great flexibility in selecting a set
of dyes having the desired spectral properties.
Dyes used in the present invention to label microparticle
populations in the different sets are selected such that the
emission spectra of each dye is detectable using the same two
detection channels, and the relative amount of emissions in each of
the two detection channels is distinguishably distinct between
different dyes. Selection of candidate dyes can be carried out
routinely based on the emission spectra of the dyes. Candidate dyes
are then evaluated empirically by dyeing microparticle populations
using a concentration series of each dye and subsequently analyzing
the results. A suitable subset of the dyed microparticles are then
selected for use together in a single array.
Depending on the application, the dyes may be selected based on
additional criteria. For example, in embodiments in which the
microparticles are used in a binding assay wherein an additional
reporter fluorophore is used to measure the amount of binding, it
is advantageous to minimize the spillover of the microparticle
emissions into the channel used to measure the reporter
fluorescence. The detection channels are selected such that the two
channels used for detecting the microparticle emission are
spectrally separated from the reporter channel, and the dyes used
are selected to minimize spillover of the microparticle emissions
into the reporter channel.
Examples of fluorophores from which a suitable set can be selected
include, but are not limited to,
4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid;
acridine and derivatives such as acridine, acridine orange,
acrindine yellow, acridine red, and acridine isothiocyanate;
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate
(Lucifer Yellow VS); N-(4-amino-1-naphthyl)maleimide;
anthranilamide; Brilliant Yellow; coumarin and derivatives such as
coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine and
derivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7;
4',6-diaminidino-2-phenylindole (DAPI);
5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylaminocoumarin; diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL);
4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin
and derivatives such as eosin and eosin isothiocyanate; erythrosin
and derivatives such as erythrosin B and erythrosin isothiocyanate;
ethidium; fluorescein and derivatives such as 5-carboxyfluorescein
(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl,
naphthofluorescein, and QFITC (XRITC); fluorescamine; IR144;
IR1446; Green Fluorescent Protein (GFP); Reef Coral Fluorescent
Protein (RCFP); Lissamine.TM.; Lissamine rhodamine, Lucifer yellow;
Malachite Green isothiocyanate; 4-methylumbelliferone; ortho
cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon
Green; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and
derivatives such as pyrene, pyrene butyrate and succinimidyl
1-pyrene butyrate; Reactive Red 4 (Cibacron.TM. Brilliant Red
3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine
(ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine,
rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B,
rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B,
sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine
101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA),
tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate
(TRITC); riboflavin; rosolic acid and terbium chelate derivatives;
xanthene; or combinations thereof. Other fluorophores or
combinations thereof known to those skilled in the art may also be
used, for example those available from Molecular Probes (Eugene,
Oreg.) and Exciton (Dayton, Ohio).
It will be clear to one of skill in the art that the suitability of
particular dyes or classes of dyes will depend on the method by
which the microparticles are labeled, as described further, below.
For example, large fluorescent proteins may be suitable for
labeling microparticles by binding the dyes to the surface of the
microparticle, but likely would not be suitable for internal
labeling using bath-dyeing methods. Suitable candidate dyes can be
selected routinely based on the labeling methods used.
III. Labeling of Microparticles
Fluorescent dyes have been incorporated into uniform microspheres
in a variety of ways, for example by copolymerization of the
fluorescent dye into the microspheres during manufacture (U.S. Pat.
No. 4,609,689 to Schwartz et al. (1975), U.S. Pat. No. 4,326,008 to
Rembaum (1982), both incorporated by reference); by entrapment of
the fluorescent dye into the microspheres during the polymerization
process; or by non-covalent incorporation of the fluorescent dye
into previously prepared microspheres (U.S. Pat. Nos. 5,326,692;
5,723,218; 5,573,909; 5,786,219; and 6,514,295; each incorporated
by reference). The method of labeling the microspheres is not a
critical aspect of the invention; any method that allows the
labeling of the microparticles with a controllable amount of dye
can be used.
In a preferred embodiment, the fluorescently labeled microspheres
of the invention are prepared by bath dying of microspheres
according to well-known methods. Bath dyeing methods are described,
for example, in U.S. Pat. Nos. 5,326,692; 5,723,218; 5,573,909;
5,786,219; and 6,514,295, which describe bath dyeing methods using
a plurality of dyes, which are equally applicable to dyeing
microparticles with single dyes.
IV. Methods of Use
The microparticle arrays of the present invention can be used
essentially in any application in which multiplex microparticle
arrays are used or are useful, including application in which the
microparticles are used a solid substrates for ligand binding
assays or as labeling reagents.
A preferred use of the arrays is to implement multiplex binding
assays for the simultaneous detection of two or more analytes
using, for example, flow cytometry or microscopy. For use in such
assays, the microparticles are coated with analyte-specific
reagents such that microparticles within a population are coated
with the reagents of the same known specificity and microparticles
in different populations are coated with reagents having different
specificities. The identity of the microparticle populations,
determined from the microparticle fluorescence, enables
identification of the analyte bound to the microparticle through
the analyte-specific reagent.
Analyte detection assays can be carried out using both competitive
and non-competitive formats. Examples of non-competitive assays
include sandwich assays in which a second analyte-specific reagent
(a reporter) is labeled to facilitate detection of analytes bound
to a microparticle. The microparticle array and fluorescently
labeled reporter reagents, one for each species of analyte to be
detected, are incubated with a sample containing (or suspected of
containing) the analytes of interest to allow for the formation of
bead-analyte-reporter complexes for each analyte present. The
resulting complexes are analyzed, preferably by flow cytometry, to
identify and, optionally, quantify the analytes present in the
sample. Because the identity of the analyte bound to the complex is
indicated by the identity of the bead, multiple analytes can be
simultaneously detected using the same fluorophore for all reporter
reagents.
In a competitive assay, the sample suspected of containing analyte
is incubated with the microparticle array and an analyte-analogue
that is capable of competing with the analyte for the limited
number of binding sites provided by coated microparticle. In one
embodiment, analyte-analogue labeled with a reporter fluorophore is
provided in a concentration sufficient to saturate the binding
sites on the microparticle. The presence of analyte, which competes
with and thereby reduces the number of labeled analyte-analogues
bound to the microparticle, results in a measurable decrease in the
reporter fluorescence associated with the microparticle.
In an alternative embodiment, microparticle arrays of the present
invention are useful as labeling reagents. The microparticles in
each population are coated with an target-specific reagent, wherein
the target is any molecule to be labeled. The sample is incubated
with the array for a time sufficient to allow the target molecules
to bind to the target-specific reagent coating the microparticles,
thus labeling the molecules. The use of the multidimensional arrays
of the invention enables labeling a large number of distinct target
molecules while requiring only two detection channels to uniquely
identify the labels. In a preferred embodiment, microparticle
arrays of the present invention are used as labeling reagents in
fluorescence microscopy.
Methods for attaching an antibody or other target-specific reagent
to a microparticle are known in the art. Commercially available
microparticles typically are provided with amino groups or carboxyl
groups to facilitate the covalent attachment of antibodies using
well known chemistry. However, any method used by those skilled in
the art may be employed.
EXAMPLES
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Example 1
Preparation a Microparticle Array
This example describes preferred methods for preparing a
microparticle array for use in flow cytometry.
The array described herein is intended for use in a detection assay
in which the microparticles are coated with an analyte-specific
reagent, and a labeled-reporter reagents are used to measure the
amount of analyte bound to a microparticle. To maximize sensitivity
of the detection assay, it is desirable to label the reporter
reagents with a particularly bright fluorophore, preferably
phycoerythrin (PE), which has an emission maximum at 578 nm. Thus,
the array is prepared assuming that an appropriate detection
channel is reserved for measuring the PE-labeled reporter
reagents.
1. Detection Channels
The detection channels used to measure the microparticle
fluorescence are selected from the channels available using a flow
cytometer. Preferred flow cytometers are the BD FACSCalibur.TM.
flow cytometer and the BD FACSArray.TM. flow cytometer (BD
Biosciences, San Jose, Calif.). The filters, which define the
detector channels, differ slightly between the two instruments (in
the standard, commercially available configurations).
A BD FACSCalibur dual laser flow cytometer has 488 nm and 635 nm
excitation lasers. Fluorescence following excitation by the 488 nm
laser is measured in the following detection channels:
FL1: 530/30 nm (515-545 nm)
FL2: 585/42 nm (564-606 nm)
FL3: 670 nm Long Pass (>670 nm)
Fluorescence following excitation by the 635 nm laser is measured
in the following detection channel:
FL4: 661/16 nm (653-669 nm)
Using a BD FACSCalibur flow cytometer, the FL2 channel is used for
measuring the PE-labeled reporter, and microparticle populations
are identified from the emissions in the FL3 and FL4 detector
channels.
The BD FACSArray flow cytometer has 532 nm and 635 nm excitation
lasers. Fluorescence following excitation by the 532 nm laser is
measured in the following detection channels:
Yellow: 585/42 nm (564-606 nm)
Far Red: 685 nm long pass (>685 nm)
Fluorescence following excitation by the 635 nm laser is measured
in the following detection channels:
NIR (Near Infra Red): 780/60 nm (750-810 nm)
Red: 661/16 nm (653-669 nm)
Using a BD FACSArray flow cytometer, the Yellow detection channel
is used to measure the PE-labeled reporter reagent, and bead
populations are identified from the emissions in the Red and Far
Red detector channels.
2. Dyes
Candidate dyes are selected in a routine manner based on their
emission spectra such that the all the dyes emit in the two
detection channels selected for use in identifying bead
populations, as described above. Additionally, the dyes are
selected such that the emission of the dyes in the PE-detection
channel (FL2 or Yellow) is minimal. Preferred sets of preferred
dyes are described in the following examples.
3. Preparation of Microparticles
Suitable undyed microparticles for use in constructing an array are
7.7 micron, 10% solid polymeric beads from Bangs Laboratories
(Carmel, Ind.). Prior to dyeing, the beads are prepared as follows.
The beads are washed 3 times in 15 ml MeOH. The beads are then
washed in 15 ml of anhydrous MeOH (MgSO.sub.4-treated) and then
suspended in 17.5 ml of anhydrous MeOH. For dyeing, 1 ml portions
(3.86.times.10.sup.7 beads) are put into separate tubes, each
containing a small stirring bar, and stirred.
4. Preparation of Dyeing Solutions
For each dye, a series of 1.0 ml dyeing solutions are prepared
consisting of the dyeing reagents in the proportions shown in the
table, below.
TABLE-US-00001 Dye Tube 30% MeOH/70% EtOH 1,4 Dioxane (0.1
.mu.mole/.mu.l in DMF) A 500 .mu.l 420 .mu.l 80 .mu.l B 540 .mu.l
420 .mu.l 40 .mu.l C 560 .mu.l 420 .mu.l 20 .mu.l D 570 .mu.l 420
.mu.l 10 .mu.l E 575 .mu.l 420 .mu.l 5 .mu.l F 577.5 .mu.l 420
.mu.l 2.5 .mu.l G 578.75 .mu.l 420 .mu.l 1.25 .mu.l H 579.4 .mu.l
420 .mu.l 0.6 .mu.l
Alternatively, another suitable organic solvent, such as
CH.sub.2Cl.sub.2, can be used in place of 1,4Dioxane. 5. Bead
Dyeing
Each of the 1 ml dyeing solutions is added to a separate tube
containing a 1 ml preparation of washed beads while stirring
vigorously at room temperature. The bead/dye mixtures are stirred
at 50.degree. C. for 1 hour in the dark to allow the dye to be
absorbed into the beads.
After dyeing, 10 ml of MeOH are added to each tube. The tubes are
vortexed and then centrifuged at .about.3,000 rpm for 4 minutes to
pellet the dyed beads. The pellets are washed 2 times with 5 ml
MeOH, then washed with 10 ml of 0.05% Tween-20, 0.1% NaN.sub.3,
then suspended in 3 ml of 0.05% Tween-20, 0.1% NaN.sub.3. The
concentration of the resulting dyed bead solution is approximately
10.sup.4/.mu.l (10.sup.7/ml).
6. Testing and Selection of Bead Populations
Dyeing is carried out as described above using each of the
candidate dyes to generate a plurality of bead sets, each set dyed
with a different candidate dye, and each set containing a series of
bead populations dyed with different concentrations of the same
dye. The plurality of bead sets represents a candidate array from
which a suitable array is selected. The selection of a suitable
array is determined empirically, and will depend on a number of
parameters, such as the dyes selected and the dyeing
efficiencies.
To select an suitable array from the candidate array, bead
populations are analyzed on either a BD FACSCalibur.TM. flow
cytometer or a BD FACSArray.TM. flow cytometer using the detector
channels described above, and the fluorescence intensity data from
each bead in each of the two detector channels are plotted in a
two-dimensional dot-plot. Bead populations that form well-defined
clusters that are simultaneously on-scale (i.e., the data not
compressed against an edge of the dot-plot) and essentially not
overlapping with other bead populations are selected for use in the
combined array.
Additionally, the emission from the beads is measured in the
PE-channel (FL2 or Yellow) to determine the amount of spillover
into the channel used to measure the PE-labeled reporter molecules.
Preferably, the spillover into the PE-channel is minimal so as to
minimize interference in the measurement of the reporter
reagents.
Because the selection of bead populations to use in the array is
empirical, bead dyeing and analysis is expected to be an iterative
process in which beads are incrementally added to an existing
array. Appropriate adjustments can be made to the dyeing procedure
in a routine manner based on the experimental results. For example,
if most populations within an initially dyed set are off scale
because the dye intensity is too high, such that only a subset was
selected for use in the final array, additional populations of
beads can be dyed using a lower concentration of dye. Subsequent
analysis with the first dyed populations selected for inclusion in
the array will allow for extending the size of the array.
Furthermore, an additional set of bead populations, dyed with a
distinct dye, can be added incrementally to an existing set to
extend the size of the array.
Example 2
29-Population Array
Bead subsets were prepared essentially as described in Example 1
using each of the dyes listed in the table, below. All dyes were
obtained from Exciton (Dayton, Ohio). Excitation and emission
maxima, which were measured in either ethanol, methanol, or
dichloromethane, are known to be somewhat dependent on the solvent
used for the measurements, and slightly different results may be
obtained using different solvents.
TABLE-US-00002 Dye Excitation Max Emission Max Populations used in
array LD700 647 673 7 LDS730 614 695 4 LDS750 572 704 3 LDS751 542
700 1 Oxazine 725 645 676 6 ABS643 640 655 8
The emission spectra of these dyes (with the exception of LDS751)
are shown in FIG. 1. Also shown are the boundaries of the detection
channel defined using a BD FACSArray flow cytometer.
Sets of dyed beads, each containing bead populations dyed with
different amounts of one of the dyes, were combined and were
analyzed on a BD FACSArray flow cytometer, as described above. Bead
populations that were both uniquely distinguishable and
simultaneously on-scale in a Far Red.times.Red dot-plot were
selected empirically for use in the final multiplex bead set. FIG.
2 shows a dot-plot (Far Red.times.Red) of the selected bead array
containing 29 distinguishable bead populations. The number of bead
populations used in the final set from each of the subsets is
indicated in the table, above.
The emissions of the bead populations in the PE-channel (Yellow)
also were measured (results not shown), and minimal spillover was
observed for all populations.
Example 3
Extended Array
The bead array described in Example 2 was incrementally extended by
the inclusion of a bead set dyed using LDS765 (Exciton, Dayton,
Ohio), prepared essentially as described above. The measured
excitation and emission maxima for the dye are shown in the table,
below:
TABLE-US-00003 Dye Excitation Max Emission Max Populations used in
array LD765 595 752 3
For this analysis, the sets of dyed beads, were analyzed on a BD
FACSCalibur flow cytometer, as described above. Bead populations
from the set dyed with LDS765 were selected such that the
populations were both uniquely distinguishable and simultaneously
on-scale relative to the bead populations in the previously
constructed array. Three populations could be added to the
29-population array. FIG. 3 shows a dot-plot (FL3.times.FL4) of the
selected bead array containing 32 distinguishable bead
populations.
The emissions of the bead populations in the PE-channel (FL2) also
were measured (results not shown), and minimal spillover was
observed for all populations.
* * * * *